Increased EGFR expression induced by a novel oncogene, CUG2, confers resistance to doxorubicin through Stat1-HDAC4 signaling

Abstract

Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR. EGFR expression wasassessed using Western blotting, immunofluorescence and capacitanceassays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stablytransfected with aCUG2expression vector (A549-CUG2; BEAS-CUG2) oran empty control vector (A549-Vec; BEAS-Vec). AftersiRNA-mediated EGFR, Stat1 andHDAC4silencing, antioxidant and multidrug resistance proteinand mRNA levels wereassessedusing Western blottingand RT-PCR.In addition, the respective cells were treated with doxorubicinafter which apoptosis and reactive oxygen species (ROS)levels weremeasured.Stat1 acetylation was assessed by immunoprecipitation. We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFRsilencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR. Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.